1. Field of the Invention
The present invention relates to a pretreatment kit for saliva for identification and quantitatively determining streptococci mutans, as one of cariogenic bacteria in human saliva, by immunochromatography utilizing an antigen-antibody reaction, and a pretreatment method for saliva using the pretreatment kit for saliva.
2. Description of Conventional Art
It has been known that there is close relation between the presence of streptococci mutans and the generation of dental caries in a human mouth, and therefore, the morbidity risk and the current condition of morbidity can be comprehended to provide benefits to quite a number of people if the presence or absence and the amount of streptococci mutans in a human mouth can be conveniently examined.
An examination technique utilizing an antigen-antibody reaction in examining has been conventionally carried out. For example, the immunoenzymatic technique, which is a method of identification and quantitatively determining with coloring density using an enzyme, requires a special washing device and complicated and accurate operations for handling an antibody and a sample, and also requires an incubator for carrying out an enzyme reaction. The fluorescent antibody technique, which is a method of specifically staining an antigen that is reacted with an antibody labeled with a fluorescent dye, is not commonly employed since a fluorescent microscope is necessary as a measurement device.
Accordingly, various techniques have been proposed for conveniently utilizing an antigen-antibody reaction. Examples thereof include a measurement technique utilizing chromatography (as shown, for example, in U.S. Pat. Nos. 5,591,645, 4,855,240, 4,435,504 and 4,980,298, and Japanese Patent Application Publication Nos. JP-A-61-145459 and JP-A-6-160388). The technique is excellent in simpleness because the presence or absence and the amount of an antigen can be measured only by mixing a body fluid thus collected in a test solution containing an antigen to be identified and quantitatively determined, and then instilling in an examination device. The technique is generally referred to as an immunochromatography technique, and the principles of identification and quantitative determination have been disclosed in detail (as shown, for example, in Se-Hwan Peak, Seung-Hwa Lee, Joung-Hawan Cho and Young-Sang Kim, “Development of rapid One-Step immunochromatographic assay, Methods”, vol. 22, p. 53 to 60 (2000)).
It seems that identification and quantitative determination of streptococci mutans in the human mouth can be carried out by applying the technique, but it has not been put into practical use because of the following problems. That is, it is necessary that a sample used for the immunochromatography technique pass through a porous membrane by the capillary phenomenon. However, because the major sample applied to the examination of bacteria in the mouth, such as streptococci mutans, is saliva, a high viscosity substance present in saliva, which is referred to as mucin, clogs the pores of the porous membrane. Furthermore, because mucin has such a function that aggregates epithelial attachment cells stripped off from oral mucosa, the pores of the porous membrane are clogged with these substances to inhibit transmission of streptococci mutans.
In addition to mucin, there is another problem complicating identification and quantitative determination of streptococci mutans by the immunochromatography technique. That is, the streptococci mutans to be measured is a bacterium having a diameter of about 1 μm solely but often forms a chain with 10 to 20 or more bacteria owing to the nature of streptococci, which may be a factor of inhibiting migration in the porous membrane. Furthermore, the streptococci mutans forms glucan having adherence from sucrose in foods and is often severely aggregated. The chain formation and aggregation of streptococci mutans cause clogging in the porous membrane and also reduce the surface area of the streptococci to affect quantitative determination of the number of antigens present on the surface of the streptococci mutans, which reduces accuracy of the measurement.
In the immunochromatography technique, detection of an analytic object is generally carried out by using two antibodies. The first antibody is retained in a porous membrane formed with glass fibers or the like on the side where a sample is dropped, and the antibody is generally labeled with latex particles, gold colloid particles or the like (hereinafter, sometimes referred to as a labeled antibody). In the case where the analytic object to be measured is present in the sample, when passing the sample through the porous membrane, the labeled antibody recognizes the analytic object to be measured and is bonded thereto. The composite of the analytic object and the labeled antibody is flowed by capillary phenomenon toward another porous membrane having the second antibody (hereinafter, sometimes referred to as a trap antibody) immobilized thereon, for example, in the form of strips, and the complex of the analytic object and the labeled antibody is recognized, trapped and detected as a visible signal (in the form of strips in this case). In the case where the immunochromatography technique is applied to saliva as a sample, however, the composite of a labeled antibody and streptococci mutans is trapped in the membrane retaining the labeled antibody but does not efficiently flow by capillary phenomenon toward the porous membrane having the trap antibody immobilized therein to cause such a problem that the accuracy of the measurement is reduced.